Various methods may be used in biology and in medicine to observe different targets in a biological sample. For example, analysis of proteins in histological sections and other cytological preparations may be performed using the techniques of histochemistry, immunohistochemistry (IHC), or immunofluorescence. Analysis of proteins in biological samples may also be performed using solid-state immunoassays, for example, using the techniques of western blots, or using cell-based assays that can be performed, for example, by using flow cytometry.
Many of the current techniques may detect only a few targets at one time (such as IHC or fluorescence-based Western blots where number of targets detectable is limited by the fluorescence-based detection system) in a single sample. Further analysis of targets may require use of additional biological samples from the source, limiting the ability to determine relative characteristics of the targets such as the presence, absence, concentration, and/or the spatial distribution of multiple biological targets in the biological sample. Moreover, in certain instances, a limited amount of sample may be available for analysis or the individual sample may require further analysis.
Methods of iteratively analyzing an individual sample are described in U.S. Pat. No. 7,629,125 and U.S. Pat. No. 7,741,046. In particular, U.S. Pat. No. 7,741,046 provides methods of detecting multiple targets in a biological sample that involve the use of oxidation for inactivating signal generators (e.g., for bleaching fluorescent dyes.) The oxidation reaction is accomplished by using oxidizing reagents, such as hydrogen peroxide. Furthermore U.S. Pat. No. 8,568,991 issued Oct. 29, 2013 and U.S. patent application Ser. No. 13/786,747 filed Mar. 6, 2013 shows, a signal can be inactivated by continuous exposure of the signal generator to irradiation, i.e., by photobleaching using anionic borates having a cationic counter ion.
However anionic borates, with a cationic counter ion, while they perform well, are not as efficient as desired for probes with a xanthene class of dyes. They also show high susceptibility to oxidation and often require special storage and handling protocols. As such a class of borates which are stable to oxidation and which can perform in a similar fashion in iterative staining especially to rapidly bleach xanthene dyes is desirable.